γδ T cells mediate robust anti‐HIV functions during antiretroviral therapy regardless of immune checkpoint expression

Abstract Objectives Although antiretroviral therapy (ART) efficiently suppresses HIV viral load, immune dysregulation and dysfunction persist in people living with HIV (PLWH). γδ T cells are functionally impaired during untreated HIV infection, but the extent to which they are reconstituted upon ART is currently unclear. Methods Utilising a cohort of ART‐treated PLWH, we assessed the frequency and phenotype, characterised in vitro functional responses and defined the impact of immune checkpoint marker expression on effector functions of both Vδ1 and Vδ2 T cells. We additionally explore the in vitro expansion of Vδ2 T cells from PLWH on ART and the mechanisms by which such expanded cells may sense and kill HIV‐infected targets. Results A matured NK cell‐like phenotype was observed for Vδ1 T cells among 25 ART‐treated individuals (PLWH/ART) studied compared to 17 HIV‐uninfected controls, with heightened expression of 2B4, CD160, TIGIT and Tim‐3. Despite persistent phenotypic perturbations, Vδ1 T cells from PLWH/ART exhibited strong CD16‐mediated activation and degranulation, which were suppressed upon Tim‐3 and TIGIT crosslinking. Vδ2 T cell degranulation responses to the phosphoantigen (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl pyrophosphate at concentrations up to 2 ng mL−1 were significantly impaired in an immune checkpoint‐independent manner among ART‐treated participants. Nonetheless, expanded Vδ2 T cells from PLWH/ART retained potent anti‐HIV effector functions, with the NKG2D receptor contributing substantially to the elimination of infected cells. Conclusion Our findings highlight that although significant perturbations remain within the γδ T cell compartment throughout ART‐treated HIV, both subsets retain the capacity for robust anti‐HIV effector functions.


INTRODUCTION
For people living with HIV-1 (PLWH), antiretroviral therapy (ART) efficiently suppresses viral replication and improves immunodeficiency. 1,2Interruption of treatment results in rapid viral rebound from a reservoir of long-lived provirus-harbouring cells. 3,4he necessity for lifelong adherence to ART holds considerable financial and health-associated effects. 5[10][11][12] One well-described impact of acute HIV-1 (HIV herein) infection is the substantial alteration of the composition and phenotype of unconventional T cells, including cd T cells.cd T cells exhibit MHC-independent reactivity to non-peptide antigens, and in humans are classified into two major subsets by Vd-chain usage.While the Vd1 subset is more frequent at mucosal sites, 13,14 the Vd2 subset makes up to 90% of the total cd T cell population within peripheral blood of healthy adults. 15][34][35][36][37][38][39] One potential mediator of cd T cell dysfunction is the expression of immune checkpoint molecules (ICMs).During chronic viral infections, persistent antigen exposure drives ICM expression on lymphocytes, including PD-1, TIGIT, Tim-3, CD160 and 2B4.While expression of these markers can reflect a state of immune exhaustion, the functional impact of ICM expression can vary across cellular subsets.][46][47][48][49][50][51][52][53] Currently, the impact of ICM expression on cd T cells remains poorly defined, both at steady-state and in the context of HIV infection. 54,55 addition to CD8 + T cells and NK cells, cd T cells are intriguing candidates for targeting HIV-infected cells in HIV cure strategies.While both cd T cell subsets play an important role in sensing HIV-infected cells, [56][57][58] the Vd2 T cell subset is a particularly interesting immunotherapeutic tool and may contribute to elimination of reactivated HIV-infected cells upon latency reversal. 57,591][62][63] The Vd1 T cell subset also holds potential for HIV immunotherapies, with expansion protocols for Delta One T (DOT) cells providing opportunities for clinical manipulation. 64d1 T cells are capable of antibody-dependent cellular cytotoxicity (ADCC) upon FccRIII (CD16) ligation, [65][66][67] suggesting Vd1 T cells could facilitate antibody-mediated killing of HIV-infected cells upon infusion of broadly neutralising antibodies (BnAbs).68 Furthermore, cytotoxic natural killer receptors (NKRs) such as NKG2C are also elevated on Vd1 T cells within HIV infection and may contribute substantially to target cell recognition.69 Strategies involving cd T cell-mediated elimination of HIV-infected cells must first address gaps in knowledge including mechanisms of infected cell recognition and the impact of ICMs on cytotoxicity pre-and post-expansion. Thereore, we assessed frequency and phenotype of Vd1 and Vd2 T cells in the context of chronic ART-suppressed HIV infection, characterised the functional capacity and defined the impact of ICM expression on effector functions of both subsets.We additionally explore the in vitro expansion of Vd2 T cells from PLWH on ART and the mechanisms by which such expanded cells may sense and kill HIV-infected targets.Findings here not only elucidate the impact of chronic infection and ART treatment on cd T cell subsets but also aid in a path towards cd T cell-based immunotherapies within chronic viral infections such as HIV.

Vd1 T cells are enriched for ICMs and markers of NK cell function in ART-suppressed PLWH
To assess the extent to which suppressive ART reconstitutes both the frequency and phenotype of cd T cells, we analysed circulating Vd1 and Vd2 T cells among a cohort of 25 PLWH on ART and 17 agematched uninfected (UI) controls (Supplementary table 1, Supplementary figure 1a, b).The subset of PLWH/ART donors used for phenotyping had been receiving ART treatment for a median 51 months, were virally suppressed (< 100 copies mL À1 ), with CD4 + T cell counts above 250 lL À1 .Despite reconstitution of CD4 T cells, PLWH exhibited persistent expansion of Vd1 T cells (median 0.6% UI; 2.3% ART, P = 0.003) and concurrent depletion of Vd2 T cells (median 1.3% UI; 0.5% ART, P = 0.006) relative to UI controls (Figure 1a and b).Consistent with previous reports, 17,35,55,70 we observed an inversion of the peripheral blood Vd2:Vd1 T cell ratio in PLWH/ART (median 2.35 UI; 0.22 ART, P < 0.0001) (Figure 1b).

Vd1 T cells exhibit an NK cell-like functional program during ART-treated HIV
Having observed persistent phenotypic changes and the presence of differentiated NK-like subsets of Vd1 T cells in the ART cohort, we next asked whether these were associated with impaired functional responses.To do so, CD3 or CD16 were crosslinked to the murine FccR expressing P815 cell line using monoclonal antibodies on a subset of 12 donors from the PLWH/ART cohort.We measured expression of CD69 and CD107a on CD27 dim/À Vd1 T cells (excluding the CD27 hi na€ ıve-like subset) (Figure 2a-d, Supplementary figure 2a,  b), confirming that both CD3 and CD16 crosslinking were sufficient to trigger both activation and degranulation relative to an isotype control.
Perturbations in Vd2 T cell memory states are partially reconstituted in ARTsuppressed PLWH We next characterised the phenotype and function of Vd2 T cells within ART-treated PLWH (Supplementary figure 1a, c).Interestingly, we observed that Tim-3 (median 2.5% UI; 13.5% ART) was the only ICM differentially expressed on Vd2  T cells among PLWH/ART in comparison to uninfected controls (Figure 3a and b).Contrastingly, we found Vd2 T cells from both UI and PLWH/ART groups expressed similarly high levels of 2B4, CD160 and PD-1, with a substantial degree of variability between donors within the two cohorts (Figure 3a), while TIGIT expression was generally minimal across both groups.The impact of chronic infection on the differentiation state of Vd2 T cells was also apparent, with those from the ART group more frequently taking on a T central memory (TCM)-like CD27 + CD45RA À phenotype (median 51.5% UI; 68.9% ART), and less frequently exhibiting a T na€ ıve (Tn)-like CD27 + CD45RA + phenotype (median 22.4% UI; 13.3% ART) (Figure 3c and d).Given the significantly elevated expression of Tim-3 among the ART cohort, we assessed its expression across memory subsets.Tim-3 expression was elevated on Tn (median 3.4 UI; 10.6% ART, P = 0.003), TCM (median 1.1% UI; 6.9% ART, P < 0.0001) and TEM-like (median 2.9% UI; 16.9% ART, P = ns/0.055)Vd2 T cell subsets in PLWH/ART compared to uninfected controls, with the most pronounced expression on TEMRA-like CD27 À CD45RA + cells (median 7.3% UI; 42.3% ART, P = 0.002) (Figure 3e).

Vd2 T cells from ART-treated PLWH exhibit impaired sensitivity to low-dose HMB-PP stimulation
To investigate whether the residual Vd2 population was functionally competent in the PLWH/ART group, we stimulated PBMC from a subset of eight PLWH/ART patients with the potent bacterial phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) and compared responses with age-matched healthy controls (Supplementary figure 4a-c).Activation (CD69) and degranulation (CD107a) were measured after 5 h of in vitro stimulation and varied substantially across individuals (Figure 4a-c).Nonetheless, Vd2 T cell responses among the ART cohort were negligible at the lowest HMB-PP dose tested (0.02 ng mL À1 ), with only three out of the eight individuals exhibiting responses above background (Figure 4b  and c).This stands in contrast to the healthy control donors, where 100% of participants exhibited responses.Furthermore, degranulation of Vd2 T cells was significantly reduced in the ART cohort at HMB-PP concentrations up to 2 ng mL À1 (median 23.8% UI vs. 7.1% ART, P = 0.021; Figure 4b), with incomplete restoration even at 20 ng mL À1 (median 24.8% UI; 9.7% ART, P = ns/0.105;Figure 4b).Interestingly, Vd2 T cells from PLWH/ART were not fully refractory to activation, as HMB-PP-induced CD69 expression was more comparable between groups (Figure 4c), although there was a trend towards lower activation for the PLWH/ART group.
As 2B4, CD160, PD-1 and Tim-3 were all expressed on Vd2 T cells within the cohort of PLWH on ART, we assessed whether inhibitory signalling through these receptors could be contributing to impaired HMB-PP responsiveness.HMB-PP-mediated Vd2 responsiveness was not impacted by blocking with monoclonal antibodies against any of the ICMs, with no significant changes in expression of either CD69 or CD107a (Figure 4d-g).Therefore, we conclude that it is unlikely that the impaired responsiveness to HMB-PP ex vivo observed in PLWH/ART is because of differential expression of ICMs between these cohorts.

Vd2 T cells from ART-treated PLWH exhibit a reduced capacity for in vitro expansion
In vitro or in vivo-expanded Vd2 T cells are a potential immunotherapeutic tool for the treatment of a number of infectious diseases.In line with this, we assessed whether ART-suppressed chronic HIV infection impacts the expansion potential of Vd2 T cells, and whether such expanded cells were capable of targeting HIV-infected cells.PBMC from a subset of 16 PLWH/ART were treated with zoledronate plus IL-2 to induce in vitro expansion of Vd2 T cells.
The cellular composition of cultures was assessed by flow cytometry on day 10 or 11 (Supplementary figure 5a).Vd2 expansion was poor in nine out of the 16 PLWH/ART, where Vd2 T cells composed < 60% of the resultant culture (Figure 5a).In contrast, expansion from healthy donors which resulted in cultures with median frequencies of about 80-92.1% Vd2 + CD3 + (Supplementary figure 5b). 73Contaminating cells in expansions from PLWH/ART were largely ab T cells (median 18.2%) and CD3 À CD56 + NK cells (median 10.6%) (Figure 5b).The best correlate of Vd2 expansion for PLWH/ART donors was the baseline frequency of Vd2 T cells in PBMC (Spearman's r 0.52; P = 0.05) (Figure 5c), rather than any markers of Vd2 T cell differentiation (Supplementary figure 5c, d).We next assessed the modulation of ICM expression during in vitro expansion (Supplementary figure 6a, b).Expanded Vd2 T cells exhibited near-universal up-regulation of Tim-3 (median 0.8% day 0; 97.4% day 14) coupled with near-total loss of CD160 (median 29.6% day 0; 0.3% day 14) (Supplementary figure 6b, Figure 5d and e).Neither PD-1 nor TIGIT expression were significantly modulated during expansion (Figure 5f and g).Finally, we assessed expression of NKG2D, a surface receptor known to contribute substantially to Vd2 T cellmediated cytotoxicity, which may be involved in the recognition of HIV-infected cells.The frequency of NKG2D + Vd2 T cells was significantly increased after in vitro expansion (median 73.7% day 0; 97.7% day 14, P = 0.010) (Figure 5h and i).

Expanded Vd2 T cells from ART-treated PLWH maintain efficient anti-HIV effector functions
To assess whether expanded Vd2 T cells maintain anti-HIV effector functions in ART-suppressed chronic HIV infection, we performed infected cell elimination (ICE) assays against the 8E5/LAV cell line, a CEM-derived cell line that contains a single copy of the HIV provirus. 74,75Importantly, cultures of 8E5 cells contain a mix of provirus transcribing cells and cells that have lost the ability to produce viral antigens.Detection of the p24 antigen via flow cytometry allows identification of HIV transcribing cells in such mixed cultures. 76Lysis of p24 + (HIV antigen expressing) or p24 À (non-antigen expressing) 8E5 cells was measured after a 4-h co-incubation with expanded Vd2 T cells at effector to target (E:T) cell ratios of 5:1.2:1, 1:1, 1:2, 1:5 and 1:10 (Supplementary figure 7a).Expanded Vd2 T cell cultures from PLWH/ART containing less than 70% Vd2 T cells were depleted of contaminating ab and/or Vd1 T cells prior to use in ICE assays (Supplementary figure 7b).
Despite the persistent defects in ex vivo responsiveness to HMB-PP, Vd2 T cells from the ART group efficiently eliminated HIV-infected cells (Figure 6a).At the highest E:T ratio tested (5:1), expanded Vd2 T cells from uninfected donors demonstrated considerable cytotoxicity against infected (p24 + ) cells (median 80.6%; Figure 6b).Vd2 T cells expanded from PLWH/ART also displayed substantial elimination of p24 + 8E5 cells (median 94.4%; Figure 6a).Impressively, Vd2 T cells expanded from PLWH/ART were still able to efficiently kill infected cells at the 1:10 E:T ratio (median 16.0%).Furthermore, p24 + cells were preferentially killed, with a lower level of cytotoxicity against the p24 À 8E5 cells, and minimal killing of the uninfected parental like CEM.NKR CCR5 cell line (Supplementary figure 7c).These data indicate HIV infection of this cell line is likely contributing to recognition by Vd2 T cells, which is further elevated upon transcription of viral antigens.
To determine the mechanism of infected cell elimination, we first examined expression of ligands for the key Vd2 T cell cytotoxic surface receptors DNAM-1, 2B4 and NKG2D on 8E5 target cells using Fc-chimera proteins (Supplementary figure 8a).As the ligands for 2B4 and NKG2D, but not DNAM-1, were expressed to high degrees on target cells (Supplementary figure 8b-d), we next blocked 2B4 or NKG2D within the infected cell elimination assay at E:T ratios of 1:1.Here, we observed that blocking NKG2D significantly diminished the targeting of both p24 + (median 24.79% decrease UI, median 58.36% decrease ART) and p24 À (median 15.94% decrease UI, median 46.35% decrease ART) cells by expanded Vd2 T cells when compared to an isotype control (Figure 6c and d), suggesting the NKG2D surface receptor contributes substantially to recognition of HIV-infected cells by expanded Vd2 T cells.

DISCUSSION
Although ART efficiently suppresses viral replication and reconstitutes CD4 T cell counts, ongoing inflammation and incomplete restoration of immune function results in persistently elevated risk of co-morbidities and co-infections such as TB. 77,78he extent to which perturbations within the cd T cell compartment are restored following ART is understudied, despite the potential importance of cd T cells as an immunotherapy tool.Here, we report persistence of highly differentiated Vd1 T cells despite ART-mediated viral suppression, with elevated expression of ICMs such as Tim-3 and TIGIT that were found to suppress effector functions.Vd2 T cell reactivity to phosphoantigen stimulation remained diminished within ART-treated individuals; however, no link between ICM expression and reduced responsiveness could be identified.6][87] Notably, Fausther-Bovendo et al. (2008) reported a loss of NKG2A and acquisition of NKG2C expression on Vd1 T cells from untreated PLWH, finding NKG2C to contribute substantially to Vd1-mediated elimination of HIV-infected CD4 + T cells. 69Similar observations have been reported in elite controller (EC) cohorts, 82,85 where PLWH maintain low or undetectable plasma viraemia but experience persistent viral replication in, and damage to, the GALT. 88Our data highlight that, similar to both untreated HIV infection and EC cohorts, PLWH on ART exhibit the persistence of highly differentiated, TEMRA-like Vd1 T cells, and demonstrate that the phenotype of these highly differentiated Vd1 T cells mirrors that of matured CD16 + NKG2C + and CD16 + NKG2C + CD57 + NK cell subsets.Similar populations of expanded, cytotoxic Vd1 T cell subsets with elevated expression of CD16, CD57 and NKG2C have been described in HCMV infection, [20][21][22][23] which is highly prevalent among PLWH (90-100% seropositivity). 89,90We therefore speculate that the Vd1 expansion and differentiation observed during ART is likely to be driven by a combination of HCMV, microbial translocation and inflammatory signals, all of which persist despite effective viral suppression. 79,80,91d1 T cells nonetheless appear to remain highly functional during ART, as demonstrated by the robust CD3-and CD16-driven activation and degranulation observed in our assays, which is consistent with other reports describing the cytotoxic capacity of Vd1 T cells from both treated and untreated HIV infection.69,92 Data regarding ICM expression and function on cd T cells is sparse and often conflicting; increased expression of PD-1 on Vd1 and Vd2 T cells has been observed in ART-treated HIV infection, 55 while others report elevated expression of Tim-3, TIGIT and CD160.54 Here, we find that 2B4, CD160, Tim-3 and TIGIT were expressed by a higher proportion of Vd1 T cells in PLWH/ART compared to uninfected controls.In contrast to a previous study, 55 we observed lower PD-1 expression on Vd1 T cells in PLWH/ART compared to age-matched uninfected controls.These discrepant results may reflect baseline immunological differences in Vd1 T cells or PD-1 expression between the populations studied.Tim-3 and TIGIT were found to suppress CD16-driven effector functions, indicating a potential inhibitory role for these receptors on Vd1 T cells.Conversely, we found no evidence of inhibition of CD16-mediated activation or degranulation by PD-1 or CD160.These data, together with the heightened proportions of Vd1 T cells within both acute and ART-treated HIV infection, highlight the potential for the engagement of this subset alongside cocktails of broadly neutralising antibodies (BnAbs) to facilitate ADCC of reactivated HIV-infected cells.68 Additionally, our data suggest that blockade of Tim-3 or TIGIT, or co-stimulation through NKRs such as NKG2C or 2B4, could enhance CD16mediated effector functions. Furthr investigations to characterise the potential of Vd1 T cell-mediated ADCC of HIV-infected cells, and the utilisation of DOT cells in this context could aid in the search for a functional cure for HIV upon latency reversal.
[28][29][30][31] In parallel, increased proportions of terminally differentiated TEMRA-like Vd2 T cells have been commonly observed within untreated HIV infection. 17,25,35Several previous studies have assessed memory subset distribution within ART-treated individuals but report contrasting results regarding the impact of infection on proportions of na€ ıve, central memory or TEMRA populations. 17,35,38,55In the present study, we found elevated proportions of TCM-like and a decreased frequency of na€ ıve-like Vd2 T cells in PLWH/ART compared to age-matched uninfected individuals, while frequencies of TEMRA-like and TEM-like Vd2 T cells were similar between groups.
Overall, most evidence suggests that ART treatment may reconstitute proportions of TEMRA-like Vd2 T cells; however, perturbations evidently persist within other memory subsets, with a high degree of variation seen between study cohorts, perhaps caused by geographical location, ART regimes or differences in cohort demographics.
9]55 Blocking ICMs failed to restore sensitivity to lowdose HMB-PP stimulation, and together with our observation that Vd2 T cells in the PLWH/ART cohort did not express elevated levels of most ICMs, we conclude that these markers are unlikely to play a significant role in Vd2 T cell functions in this context.Of note, Tim-3 expression was found to be significantly heightened on Vd2 T cells within PLWH/ART across all memory states and was additionally upregulated upon in vitro expansion.As we were unable to find evidence that Tim-3 suppressed HMB-PP-mediated activation, the role of this marker on Vd2 T cells remains unclear and should be investigated within future studies.Alternatively, compromised phosphoantigen signalling to Vd2 T cells from HIV-infected APCs may contribute to this functional impairment. 93,94As Vd2 T cell phosphoantigen responsiveness is mediated through BTN3A1 and BTN2A1, 95,96 future studies should investigate the impact of acute, chronic and ART-treated HIV infection on the expression of and signalling through these molecules.
Although it was possible to expand Vd2 T cells in vitro from some donors within the PLWH/ART cohort, expansion was not as reliable or efficient as from uninfected donors.Vd2 T cell frequencies ex vivo correlated with the success of expansion.This finding could be particularly relevant in the context of therapeutic manipulation of Vd2 T cells in cohorts of ART-treated PLWH.Identification of individuals with efficient Vd2 T cell expansion capacity will inform choices regarding the use of autologous versus allogeneic immunotherapeutic approaches.Despite the high degree of variability in HMB-PP and/or zoledronate + IL-2 responsiveness, successfully expanded Vd2 T cell cultures from PLWH/ART were capable of remarkably potent effector functions against an HIV-infected cell line, with preferential killing of infected cells expressing HIV antigens.Furthermore, we identify NKG2D-mediated recognition as a key pathway for elimination of HIV-infected cells.HIV infection of primary CD4 + T cells is known to drive upregulation of the UL16-binding proteins-1 to -3, which are key ligands for NKG2D. 97Here, we detected high frequencies of NKG2D expressing Vd2 T cells from PLWH/ART, which was further increased upon in vitro expansion.We conclude that although Vd2 T cell TCR/phosphoantigen signalling pathways appear to be compromised in the context of HIV/ART, NKG2D-mediated recognition and activation is not functionally impaired, allowing for efficient elimination of infected cells.
In summary, we explored functional perturbations of cd T cell subsets in PLWH undergoing suppressive ART.We identify that the Vd1 T cell subset remains highly differentiated throughout treatment, taking on an NK cell-like phenotype with increased expression of Tim-3 and TIGIT that were found to slightly inhibit effector functions upon crosslinking.Perturbations in Vd2 T cell memory phenotypes were partially restored upon effective viral suppression, though phosphoantigen sensitivity was still significantly impaired.Despite persistent phenotypical alterations in ART-treated individuals, both cd T cell subsets maintained robust anti-HIV effector functions, illuminating a pathway towards the inclusion of cd T cell-based approaches within HIV immunotherapies.

Sample collection and isolation of PBMC from whole blood
Whole blood was collected from a total of 52 people living with HIV undergoing suppressive antiretroviral therapy (PLWH/ART) recruited through the Melbourne Sexual Health Centre between 2012 and 2023.A total of 29 uninfected controls (UI) were recruited at the University of Melbourne.PBMC were isolated from whole blood using Ficoll-Paque gradient density centrifugation (Cytiva, Cambridge, USA) and either used immediately or cryopreserved in freeze solution (90% fetal calf serum (FCS)) (Sigma-Aldrich, St. Louis, USA) and 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich) for future use.
After surface staining, cells were washed and resuspended in PBS containing 2% FCS before acquisition on a BD LSR Fortessa using BD FACS Diva.For each memory subset, donors with less than 100 events were excluded from analysis.

Flow cytometry-based infected cell elimination assay
Lysis of the 8E5/LAV HIV-infected cell line (NIH ARP-#95) was quantified using a modified version of a flow cytometry-based infected cell elimination assay previously described. 74Expanded Vd2 T cells were collected for use on day 12 or 13 of in vitro expansion.For surface receptor blocking, expanded Vd2 T cells were preincubated with 5 lg mL À1 of anti-NKG2D (1D11; Biolegend), anti-2B4 (eBioPP35; eBioscience) or an IgG1 j isotype control (MOPC-21; Biolegend) for 30 min at 37°C 5% CO 2 .8E5 target cells were stained with eFluor 670 dye (eBioscience) and added to tubes containing expanded Vd2 T cells at effector:target cell ratios of 5:1, 2:1, 1:1, 1:2, 1:5 and 1:10, or to a tube without effector cells to measure background death.For some experiments, eFluor 670 stained CEM.NKr-CCR5 cells (NIH ARP-4376) were used in place of 8E5 cells.Tubes were centrifuged at 300 9 g for 1 min, then incubated at 37°C with 5% CO 2 for 4 h.After the incubation period, eGFP-CEM.NKr cells (NIH ARP-11698) were added as a reference population to calculate elimination of p24 + or p24 À 8E5 cells.Cells were stained with Aqua viability dye (ThermoFisher), then permeabilised with Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) prior to staining with HIV p24 RD1 (KC57; Beckman Coulter, Mount Waverley, Australia).After intracellular staining, samples were washed and resuspended in PBS containing 2% FCS before acquisition on a BD LSR Fortessa using BD FACS Diva, with a consistent number of eGFP + CEM cells collected per tube.Percent cytolysis was calculated with the following formula: ([%p24 + compared to eGFP + target alone À %p24 + compared to eGFP + experimental tube] Ä %p24 + compared to eGFP + target alone) 9 100.

Expression of ligands on target cells
To assess the expression of ligands on the 8E5 cell line, cells were incubated with 5 lg mL À1 of NKG2D-Fc fusion protein (R&D Systems), 2B4-Fc fusion protein (R&D Systems), DNAM-1-Fc fusion protein (R&D Systems) or left unstained for 30 min, washed twice, then stained with Aqua viability dye (ThermoFisher).Binding of Fc-fusion proteins was detected with an APC-conjugated goat anti-human IgG antibody (HP6017; Biolegend).Samples were washed twice and then permeabilised with Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) prior to staining with HIV p24 RD1 (KC57; Beckman Coulter).After intracellular staining, samples were washed and resuspended in PBS containing 2% FCS before acquisition on a BD LSR Fortessa using BD FACS Diva.

Statistics
Flow cytometry data were analysed in FlowJo v10.2 (FlowJo, LLC, Ashland, USA).Statistical analyses were carried out using GraphPad Prism v10 (GraphPad, Boston, USA).Correlations were calculated using Spearman's r-test with two-tailed post-tests.Wilcoxon matched-pairs signed rank tests were performed for paired analysis.For unpaired data, Mann-Whitney U-tests were performed.For all t-tests, P-values < 0.05 were determined to be significant, otherwise ns.

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2024 The Authors.Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.

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2024 The Authors.Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.
phenotypes, both Vd1 and Vd2 T cell subsets from PLWH on ART exhibited a strong capacity for anti-HIV effector functions, highlighting their potential for use within future immunotherapies or HIV curative strategies.

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2024 The Authors.Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc. 2024 | Vol. 13 | e1486 Page 13